MN/CA IX/CA9 and renal cancer prognosis

ABSTRACT

Herein disclosed are methods that are prognostic for renal cell carcinoma, particularly renal clear cell carcinoma, afflicting a vertebrate. An exemplary prognostic method comprises detecting the presence of, and quantitating the level and/or extent of a MN/CA9 gene expression product in a sample from the affected subject, wherein if 50% or fewer cells are found to express the MN/CA9 gene, then the subject is considered to have a poorer prognosis. MN/CA9 gene expression products useful in the prognostic methods include MN protein, MN polypeptide and/or MN nucleic acids. The methods are useful as an aid in the selection of treatment for a patient with renal cell carcinoma, alone or in combination with conventional tumor stage and/or grade information. The methods of the invention can be used, for example, to identify those patients requiring more aggressive therapy regimens, or those patients most likely to respond to adjuvant immunotherapies, particularly MN/CA IX/CA9-targeted therapies.

FIELD OF THE INVENTION

The present invention is in the general area of medical genetics and inthe fields of biochemical engineering, immunochemistry and oncology.More specifically, it relates to the MN gene—a cellular gene consideredto be an oncogene, known alternatively as MN/CA9, CA9, or carbonicanhydrase 9, which gene encodes the oncoprotein now known alternativelyas the MN protein, the MN/CA IX isoenzyme, MN/CA IX, carbonic anhydraseIX, CA IX, the MN/G250 or the G250 protein.

More specifically, the instant invention is directed to theidentification of MN antigen or MN gene expression in renal cancerpatient samples, which provides the basis for prognostic assays forrenal cancer, particularly renal clear cell carcinoma, and for makingclinical decisions on cancer treatment.

BACKGROUND

As indicated above, the MN gene and protein are known by a number ofalternative names, which names are used herein interchangeably. The MNprotein was found to bind zinc and have carbonic anhydrase (CA) activityand is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CAIX or CA IX [22]. According to the carbonic anhydrase nomenclature,human CA isoenzymes are written in capital roman letters and numbers,whereas their genes are written in italic letters and arabic numbers.Alternatively, “MN” is used herein to refer either to carbonic anhydraseisoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydraseisoenzyme 9 (CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated bythe context.

The MN protein has also been identified with the G250 antigen. Uemura etal. [23] states: “Sequence analysis and database searching revealed thatG250 antigen is identical to MN, a human tumor-associated antigenidentified in cervical carcinoma (Pastorek et al., 1994).”

Zavada et al., International Publication No. WO 93/18152 (published Sep.16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995) describe thediscovery of the MN gene and protein. The MN gene was found to bepresent in the chromosomal DNA of all vertebrates tested, and itsexpression to be strongly correlated with tumorigenicity. In general,oncogenesis may be signified by the abnormal expression of CA IXprotein. For example, oncogenesis may be signified: (1) when CA IXprotein is present in a tissue which normally does not express CA IXprotein to any significant degree; (2) when CA IX protein is absent froma tissue that normally expresses it; (3) when CA9 gene expression is ata significantly increased level, or at a significantly reduced levelfrom that normally expressed in a tissue; or (4) when CA IX protein isexpressed in an abnormal location within a cell. WO 93/18152 furtherdiscloses, among other MN-related inventions, MN/CA IX-specificmonoclonal antibodies (MAbs), including the M75 MAb and the VU-M75hybridoma that secretes the M75 MAb. The M75 MAb specifically binds toimmunodominant epitopes on the proteoglycan (PG) domain of the MN/CA IXproteins.

Zavada et al., International Publication No. WO 95/34650 (published Dec.21, 1995) provides in FIG. 1 the nucleotide sequences for a full-lengthMN cDNA [SEQ ID NO: 1] clone isolated as described therein, and theamino acid sequence [SEQ ID NO: 2] encoded by that MN cDNA. WO 95/34650also provides in FIG. 6 the nucleotide sequence for the MN promoter.Those MN cDNA, promoter and amino acid sequences are incorporated byreference herein.

Zavada et al., International Publication No. WO 03/100029 (publishedDec. 4, 2003) discloses among other MN-related inventions, MN/CAIX-specific MAbs that are directed to non-immunodominant epitopes,including those on the carbonic anhydrase (CA) domain of the MN/CA IXprotein. An example of such a MN/CA IX-specific MAb is the V/10 MAb,secreted from the V/10-VU hybridoma The MN protein is now considered tobe the first tumor-associated carbonic anhydrase isoenzyme that has beendescribed. The carbonic anhydrase family (CA) includes elevencatalytically active zinc metalloenzymes involved in the reversiblehydration-dehydration of carbon dioxide: CO₂+H₂0

HCO₃ ⁻+H⁺. CAs are widely distributed in different living organisms. TheCAs participate in a variety of physiological and biological processesand show remarkable diversity in tissue distribution, subcellularlocalization, and biological functions [24, 25, 26]. Carbonic anhydraseIX, CA IX, is one of the most recently identified isoenzymes [22, 27].Because of the CA IX overexpression in transformed cell lines and inseveral human malignancies, it has been recognized as a tumor-associatedantigen and linked to the development of human cancers [8, 16, 28].

CA IX is a glycosylated transmembrane CA isoform with a uniqueN-terminal proteoglycan-like extension [22]. Through transfectionstudies it has been demonstrated that CA IX can induce thetransformation of 3T3 cells [22]. Recent studies have revealed that CAIX not only participates in cell adhesion, but also can be induced inhypoxia via the HIF-1 protein binding to the hypoxia-responsive elementof the MN promoter [29, 30]. The transcription of the MN gene isnegatively regulated by wild-type von Hippel-Lindau tumor suppressorgene in renal cell carcinoma cells [31]. The protein product of the vonHippel-Lindau tumor suppressor gene interacts with the ubiquitin ligasecomplex that is responsible for targeting HIF-1α for oxygen-dependentproteolysis [32, 33]. Thus, low levels of oxygen lead to stabilizationof HIF-1α, which in turn leads to the increased expression of MN [30].Areas of high expression of MN in cancers are linked to tumor hypoxia asreported in many cancers, and incubation of tumor cells under hypoxicconditions leads to the induction of MN expression [30, 34-38].

Many studies, using the MN-specific monoclonal antibody (MAb) M75, haveconfirmed the diagnostic/prognostic utility of MN indiagnosing/prognosing precancerous and cancerous cervical lesions [16,39, 40]. Immunohistochemical studies with the M75 MAb of cervicalcarcinomas and a PCR-based (RT-PCR) survey of renal cell carcinomas haveidentified MN expression as closely associated with those cancers andconfirm MN's utility as a tumor biomarker [16, 39, 41]. In variouscancers (notably uterine cervical, ovarian, endometrial, renal, bladder,breast, colorectal, lung, esophageal, head and neck and prostatecancers, among others), CA IX expression is increased and has beencorrelated with the microvessel density and the levels of hypoxia insome tumors [34, 35].

In tissues that normally do not express MN protein, CA IX positivity isconsidered to be diagnostic for preneoplastic/neoplastic diseases, suchas, lung, breast and cervical precancers/cancers [36-38], among otherprecancers/cancers. Very few normal tissues have been found to expressMN protein to any significant degree; those MN-expressing normal tissuesinclude the human gastric mucosa and gallbladder epithelium, and someother normal tissues of the alimentary tract [42-44].

Renal cell carcinoma (RCC), which accounts for 3% of all adultmalignancies, is the most lethal of the urologic cancers [1]. RCC is, inthe US, the ninth leading cause of cancer mortality, with 35,000 newcases and more than 12,000 deaths predicted in 2004 [2]. The incidenceof RCC has increased since the 1970s, largely owing to a more prevalentuse of ultrasonography and computerized tomography for the evaluation ofa variety of abdominal and gastrointestinal complaints [3]. For RCC, thebest available prognostic indicator is stage, but the current prognosticfactors: Fuhrman grade, and performance status, as well as stage, areinsufficient to predict patient outcome and cancer aggressiveness [4-6].Identification of biomarkers that provide further prognostic informationwould thus be vital for defining optimal treatment and outcomes.

As indicated above, previous studies have shown that MN, a member of thecarbonic anhydrase family, is induced constitutively in certain tumortypes but is absent in most normal tissues [7-10]. Furthermore, previousimmunobiochemical studies of malignant and benign renal tissues revealedthat MN is also highly expressed in RCC, suggesting that MN expressionis a useful diagnostic biomarker [11, 12, 15-17]. Bui et al. [17] statethat another biomarker Ki67 when used with CA IX in RCC highly predictssurvival. In addition, Zavada et al. [18] discovered a soluble form ofCA IX in the body fluids (urine and CA IX serum) of RCC patients. [Seealso, Zavada et al., International Publication No. WO 03/100029(published Dec. 4, 2003).]

Disclosed herein are methods wherein MN expression is shown to be usefulas a prognostic marker for RCC, and particularly renal clear cellcarcinoma (CCC). CCCs comprise up to about 85% of RCCs. The experimentsdisclosed herein support the promising significance of MN as a molecularmarker in RCCs, and particularly CCCs. Low MN expression in RCCs,particularly CCCs, was found to be a poor prognostic factor, andconversely high MN expression was found to be a good prognostic factor.MN expression is disclosed herein to be the best prognostic factor whencompared with T stage and Fuhrman grade. Decreased MN expression isdisclosed herein to be independently associated with poor survival.

The prognostic methods of this invention use 50% MN/CA IX/CA9 expressionas the cut-off between better and poorer prognoses for RCCs/CCCs. Liaoet al. [12] (at page 2828) described MN/CA IX immunostaining patterns“as diffuse when >50% of the cells stained and focal when ≦50% of thecells stained.” Whereas the instant inventive methods use 50% as thecut-off value, Bui et al. [15] (at page 4) found by survival treeanalysis of MN/CA IX immunostaining scoring information from tissuearrays “that a staining percentage of 85% was an ideal cutoff forstratification for patient survival.” [See also, Bui et al.International Publication No. WO 03/089659 (published Oct. 30, 2003).]

The prognostic methods of this invention can be used to predict clinicaloutcome and tumor behavior. The prognostic methods disclosed hereindetect and/or quantitate levels, extent and/or intensity of MNexpression, and can identify high-risk RCC/CCC patients who couldbenefit from adjuvant immunotherapy and MN/CA IX/CA9-targeted therapies,among other appropriate therapies.

Preliminary data from the Bui et al. [15] study indicate a relationshipbetween MN/CA IX and immunotherapy response. Similarly, therapies basedon monoclonal antibodies to MN/CA IX or immunotherapy with MN/CAIX-based RCC vaccine [15, 19-21], as well as vectors that encode acytotoxic protein/polypeptide and/or cytokine operatively linked to theMN gene promoter or a MN promoter fragment having promoter activity [asdisclosed, for example, in Zavada et al., International Publication No.WO 00/24913 (published May 4, 2000)], preferably a MN promoter fragmentcomprising a hypoxia responsive element (HRE), preferably the MN HRE,can also be considered according to the level and/or extent of MN/CAIX/CA9 expression in a RCC/CCC patient sample.

SUMMARY OF THE INVENTION

The present invention relates to methods for detecting and/orquantitating levels and/or extent of MN/CA9 gene expression products ina sample taken from a patient afflicted with renal cell carcinoma (RCC),particularly from a patient afflicted with renal clear cell carcinoma(CCC), wherein said detecting and/or quantitating is useful indetermining the prognosis of the patient. The RCC/CCC patient can be avertebrate, preferably a mammal, and more preferably a human. Themethods comprise detecting and/or quantitating levels and/or extent ofMN/CA9 gene expression product(s) in a sample comprising neoplasticcells taken from the RCC/CCC patient, and determining that the patienthas a poorer prognosis if the level and/or extent of MN/CA9 geneexpression product(s) indicates that 50% or fewer of cells in saidsample express MN/CA9 gene expression product. A poorer prognosis can bemeasured, for example, in terms of shortened cumulative survival,increased risk of recurrence and/or increased risk of metastasis.

In a preferred embodiment of the invention, the MN/CA9 gene expressionproduct is MN/CA IX antigen, and the MN/CA IX antigen is quantitated invertebrate samples, preferably mammalian samples, more preferably humansamples, comprising neoplastic cells. In addition to predicting clinicaloutcome, the methods of the present invention can also identifyhigh-risk patients in need of adjuvant therapy, and/or identifycandidates for MN/CA9/CA IX-targeted therapies, among other courses oftreatment.

In one aspect, the invention concerns methods which are prognostic forrenal cell carcinoma afflicting a subject vertebrate, wherein anexemplary method comprises:

(a) detecting the presence or absence of MN/CA9 gene expression productin a sample comprising neoplastic cells taken from said vertebrate,

(b) if MN/CA9 gene expression product is present in said sample,quantitating the level and/or extent of said MN/CA9 gene expressionproduct relative to the number of cells in said sample, and

(c) determining that said subject vertebrate has a poorer prognosis ifthe level and/or extent of MN/CA9 gene expression product of steps (a)and (b) indicates that 50% or fewer of cells in said sample expressMN/CA9 gene expression product;

wherein said MN/CA9 gene expression product is encoded by a nucleotidesequence selected from the group consisting of:

(1) SEQ ID NO: 1's coding region;

(2) nucleotide sequences that hybridize under stringent hybridizationconditions of 50% formamide at 42 degree C. to complement of SEQ ID NO:1's coding region; and

(3) nucleotide sequences that differ from SEQ ID NO: 1's coding regionor from the nucleotide sequences of (2) in codon sequence due to thedegeneracy of the genetic code. SEQ ID NO: 1 is the full-length MN cDNAas disclosed in Zavada et al. WO 95/34650, supra.

Preferred assays to be used according to the methods of the invention todetect said MN/CA9 gene expression product in detecting step (a) arethose wherein said MN/CA9 gene expression product comprises an MN/CA IXprotein or MN/CA IX polypeptide, and said assays are selected from thegroup consisting of Western blots, enzyme-linked immunosorbent assays,radioimmunoassays, competition immunoassays, dual antibody sandwichassays, immunohistochemical staining assays, agglutination assays, andfluorescent immunoassays. More preferably, said MN/CA9 gene expressionproduct detecting step (a) comprises the use of immunohistochemicalstaining, and said quantitating step (b) comprises determining thepercentage of MN/CA IX immunoreactive cells and/or the intensity and/orextent of immunostaining of MN/CA IX immunoreactive cells, wherein if50% or fewer cells in said sample are immunoreactive, concluding thatsaid vertebrate has a poorer prognosis than if more than 50% of cells insaid sample are immunoreactive. Preferably, the quantitating step (b)comprises determining the percentage of MN/CA IX immunoreactive cells.Still more preferably, said detecting step (a) comprises the use of aMN/CA IX-specific monoclonal antibody, preferably the M75 MAb secretedby the hybridoma VU-M75 which has Accession No. ATCC HB 11128.

In a preferred embodiment of the invention, the MN/CA9 gene expressionproduct is CA IX antigen, and the CA IX antigen is quantitated inpreneoplastic/neoplastic vertebrate samples, preferably mammaliansamples, more preferably human samples, wherein the vertebrate, mammalor human is afflicted with RCC, particularly CCC. Such samples can be,for example, tissue specimens, tissue extracts, body fluids, cells, celllysates and cell extracts, among other samples. Preferred tissuespecimens to assay by immunohistochemical staining, for example, includecell smears, histological sections from biopsied tissues or organs, andimprint preparations among other tissue samples. Such tissue specimenscan be variously maintained, for example, they can be fresh, frozen, orformalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Preferred tissue samples areformalin-fixed, paraffin-embedded tissue samples.

An exemplary and preferred method which is prognostic for renal cellcarcinoma afflicting a subject vertebrate comprises:

(a) detecting the presence or absence of MN/CA9 gene expression productin a sample comprising neoplastic cells taken from said vertebrate, saiddetecting comprising the use of immunohistochemical staining with MN/CAIX-specific antibody to detect the presence or absence of MN/CA IXprotein in the sample;

(b) if MN/CA IX protein is present in said sample, quantitating thelevel and/or extent of said MN/CA IX protein in said sample relative tothe number of cells, comprising determining a MN/CA IX immunoreactivityscore of cells in said sample, comprising determining the percentage ofimmunoreactive cells with cell membrane staining, wherein the percentageof immunoreactive cells is assigned an immunoreactivity score with

a value of 0 if no immunoreactive cells,

a value of 1 if 50% or less immunoreactive cells, or

a value of 2 if more than 50% immunoreactive cells;

and

wherein if the immunoreactivity score of the sample is 1 or less,concluding in step (c) that said vertebrate has a poorer prognosis thanif said immunoreactivity score is 2.

In an alternative preferred embodiment, preferred assays to be usedaccording to the methods of the invention in said MN/CA9 gene expressionproduct detecting step (a) are nucleic acid-based assays, wherein saidMN/CA9 gene expression product comprises a mRNA encoding an MN/CA IXprotein or MN/CA IX polypeptide, or a cDNA complementary to mRNAencoding an MN/CA IX protein or MN/CA IX polypeptide. Preferably, saiddetecting step (a) is by in situ hybridization, Northern blotting, PCR,RT-PCR, real-time PCR, or by quantitative real-time RT-PCR.

Preferably, the renal cell carcinoma to be tested according to theprognostic methods of the invention for MN/CA9 gene expression product,is selected from the group consisting of renal clear cell carcinoma,papillary cell carcinoma and chromophobe carcinoma. More preferably, therenal cell carcinoma is renal clear cell carcinoma. Preferably saidvertebrate is a mammal, more preferably human. Still more preferably,the vertebrate is a human patient, and said renal cell carcinoma isselected from the group consisting of renal clear cell carcinoma,papillary cell carcinoma and chromophobe carcinoma. Preferably, saidrenal cell carcinoma is a renal clear cell carcinoma tumor, and saidsample is taken from said tumor and/or from a metastatic lesion derivedfrom said tumor.

Preferred prognostic methods according to the invention are thosewherein a poorer prognosis is measured in terms of shortened cumulativesurvival, increased risk of recurrence of said renal cell carcinomaand/or increased risk of metastasis. Further preferred methods are thosewherein said renal cell carcinoma comprises a tumor or a tumor and oneor more metastatic lesions derived from the tumor, and wherein a poorerprognosis is measured in terms of shortened cumulative survival,increased risk of recurrence of said neoplastic disease and/or increasedrisk of metastasis following surgical removal of the tumor, or the tumorand said one or more metastatic lesions.

Preferably, said prognostic method is used as an aid in the selection oftreatment for said renal cell carcinoma afflicting said vertebrate. Themethods of the invention can be used, for example, to identify thosesubsets of patients with the lowest survival rates in order to establishmore aggressive therapy regimens. In one embodiment of the invention,detection and quantitation of MN/CA9 gene expression products are usedin combination with conventional tumor stage and grade information todetermine patient prognosis. For example, if said renal cell carcinomain said vertebrate has a T-stage of 3 or higher and/or a Fuhrman gradeof 3 or higher, then if the detecting step (a) and the quantitating step(b) indicate that 50% or less of cells in said vertebrate sample expressMN/CA9 gene expression product, said vertebrate should be treated withmore aggressive therapy regimens.

Even if the tumor T-stage is 3 or higher and/or the Fuhrman grade is 3or higher in said vertebrate, if more than 50% of cells in said sampleexpress MN/CA9 gene expression product, said vertebrate has a betterprognosis than if 50% or less of cells in said sample express MN/CA9gene expression product, and less aggressive therapies should beselected. Conversely, even if the tumor T-stage is 2 or lower and/or theFuhrman grade is 2 or lower in said vertebrate, if 50% or less of cellsin said sample express MN/CA9 gene expression product, said vertebratehas a worse prognosis than if more than 50% of cells in said sampleexpress MN/CA9 gene expression product, and more aggressive therapiesshould be selected.

Aspects of the instant invention disclosed herein are described in moredetail below.

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ABBREVIATIONS

The following abbreviations are used herein:

aa—amino acidAEC—3-amino-9-ethylcarbazole

ATCC—American Type Culture Collection

bp—base pairsCA—carbonic anhydraseCAI—carbonic anhydrase inhibitorCCC—clear cell subtype of renal cell carcinomaCi—curieCI—confidence intervalcm—centimeterCS—cumulative survivalC-terminus—carboxyl-terminus° C.—degrees centigradeDAB—diaminobenzidine tetrahydrochlorideds—double-strandedEDTA—ethylenediaminetetraacetateELISA—enzyme-linked immunosorbent assay

Gr—Grade

HRP—horseradish peroxidaseIC—intracellularIFN—interferon (exemplary cytokine)IL-2—interleukin-2 (exemplary cytokine)kb—kilobasekbp—kilobase pairskd or kDa—kilodaltonsM—molarMAb—monoclonal antibodymin.—minute(s)mg—milligramml—millilitermM—millimolarmmol—millimolen—number of casesng—nanogramnm—nanometernM—nanomolarnt—nucleotideN-terminus—amino terminusOR—odds ratioORF—open reading framePBS—phosphate buffered salinePCR—polymerase chain reactionPG—proteoglycanpI—isoelectric pointRCC—renal cell carcinomaRT-PCR—reverse transcription polymerase chain reactionRTU—ready to useSD—standard deviationSDS—sodium dodecyl sulfate

SPSS—“Statistical Package for the Social Sciences”

SSPE—NaCl (0.18 M), sodium phosphate (0.01 M), EDTA (0.001 M)Stg—stageTM—transmembraneTris—tris (hydroxymethyl) aminomethaneμCi—microcurieμg—microgramμl—microliterμM—micromolar

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a high pattern of MN/CA IX staining in renal papillarycarcinoma [MN/CA IX×100].

FIG. 2 shows intensive membraneous staining seen in renal clear cellcarcinoma (CCC) [MN/CA IX×200].

FIG. 3 graphically shows cumulative survival (CS) of 61 CCC patientsaccording to T stage (a), tumor grade (b), and MN/CA IX expression (c).

FIG. 4 graphically shows CS of 61 CCC patients according to T stage andMN/CA IX expression.

FIG. 5 graphically shows CS of 61 CCC patients according to tumor gradeand MN/CA IX expression.

NUCLEOTIDE AND AMINO ACID SEQUENCE SYMBOLS

The following symbols are used to represent nucleotides herein:

Base Symbol Meaning A adenine C cytosine G guanine T thymine U uracil Iinosine M A or C R A or G W A or T/U S C or G Y C or T/U K G or T/U V Aor C or G H A or C or T/U D A or G or T/U B C or G or T/U N/X A or C orG or T/U

There are twenty main amino acids, each of which is specified by adifferent arrangement of three adjacent nucleotides (triplet code orcodon), and which are linked together in a specific order to form acharacteristic protein. A three-letter or one-letter convention may beused herein to identify said amino acids as follows:

3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev. Alanine Ala A Arginine ArgR Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamic Acid GluE Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV Unknown or other X

DETAILED DESCRIPTION

The invention provides methods for prognosis of renal cell carcinoma(RCC) in a patient, particularly for renal clear cell carcinoma (CCC).The methods include quantifying the level and/or extent of MN/CA9 geneexpression product, if any, present in a sample taken from a patientthat has been diagnosed with renal cell carcinoma, particularly CCC. TheMN/CA9 gene expression product can be CA IX protein, CA IX polypeptide,CA9 nucleic acids, particularly mRNA encoding a CA IX protein orpolypeptide, a cDNA corresponding to an mRNA encoding a CA IX protein orpolypeptide, or the like. The MN/CA9 gene expression product levels arequantified relative to the number of the cells in the sample, comprisingdetermining the percentage of cells that are expressing MN/CA9, and saidlevels, including absence of MN/CA IX, are correlated with a better orworse prognosis for the patient. Said MN/CA9 gene expression product ispreferably a CA IX protein or CA IX polypeptide quantitated in a sampletaken from the patient. The methods can be used, for example, to aid inthe selection of therapies, and to monitor cancer chemotherapy, tumorreappearance and metastasis. In particular, the levels of MN/CA9 geneexpression products can be used to identify high risk patients in needof adjuvant therapies, particularly those in need of more aggressivetherapies from the outset.

A preferred method of quantifying the level and/or extent of MN/CA9 geneexpression product in a patient sample is by immunohistochemicalstaining. More preferably, said MN/CA9 gene expression product detectingstep (a) is by immunohistochemical staining, and said quantitating step(b) comprises determining the percentage of immunoreactive cells and/orthe intensity or extent of immunostaining of immunoreactive cells. Stillmore preferably, said detecting step (a) comprises the use of a MN/CAIX-specific monoclonal antibody, preferably the M75 MAb secreted by thehybridoma VU-M75 which has Accession No. ATCC HB 11128 and has beendeposited under the Budapest Treaty at the American Type CultureCollection.

In an alternative preferred embodiment, preferred assays to be usedaccording to the methods of the invention in said MN/CA9 gene expressionproduct detecting and quantitating steps (a) and (b) are nucleicacid-based assays, wherein said MN/CA9 gene expression product comprisesa mRNA encoding a MN/CA IX protein or MN/CA IX polypeptide, or a cDNAcomplementary to mRNA encoding an MN/CA IX protein or MN/CA IXpolypeptide. Preferably, said detecting and quantitating steps (a) and(b) are by in situ hybridization or by Northern blotting.

Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors, freeradicals, particles, dyes and the like. Such labeled reagents may beused in a variety of well known assays, such as radioimmunoassays,enzyme immunoassays, e.g., ELISA, fluorescent immunoassays, and thelike. See for example, U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837;and 4,233,402.

In certain embodiments of the invention, the percentage of cells in asample that are expressing mRNA or cDNA that encodes a CA IX protein ora CA IX polypeptide is determined, and thereby correlated with aprognosis for a patient. Where expression of MN/CA9 mRNA or MN/CA9 cDNAis measured, CA9 mRNA or CA9 cDNA expression in 50% or less of cells inthe sample is indicative of poorer prognosis.

Additionally, methods can be used in combination; for example, both CAIX protein and CA9 mRNA expression can be assessed byimmunohistochemistry and in situ hybridization, respectively. Apreferred embodiment would be exemplified by screening tumor samplesfrom patients with renal cell carcinoma and matched samples ofnon-neoplastic renal tissues as a control. One of ordinary skill in theart using routine methods could optimize the assays of this invention todetermine where the 50% cutoff would be, or how the 50% cutoff could bedetermined for different types of tissue samples for patients with renalcell carcinoma, particularly renal clear cell carcinoma.

It can be appreciated by those of skill in the art that various otherpreneoplastic/neoplastic samples can be used to quantify the MN/CA9 geneexpression products. For example, in the case of a patient afflictedwith a renal cell carcinoma, the sample may be taken from the tumor orfrom a metastatic lesion derived from the tumor, or from theextracellular fluid within or immediately surrounding the tumor ormetastatic lesion.

It can further be appreciated that alternate methods, in addition tothose disclosed herein, can be used to detect and quantify the MN/CA9gene expression products.

Neoplastic Cells/Tissues

As used herein, “cancerous” and “neoplastic” have equivalent meanings,as well as “precancerous” and preneoplastic”.

As used herein, “renal cell carcinoma” or “RCC” is considered to be acarcinoma of the renal parenchyma, and is also called renal cancer,adenocarcinoma of the kidney, renal adenocarcinoma, hypernephroidcarcinoma, hypernephroma, or Grawitz's tumor. Renal clear cell carcinomaor “CCC” is the predominant subtype of renal cell carcinoma comprisingup to about 85% of RCCs. The other 3 subtypes of RCC are the granularcell, mixed granular and clear cell, and spindle cell subtypes.

In a preferred embodiment of the invention, the MN/CA9 gene expressionproduct is MN/CA IX antigen, and the MN/CA IX antigen is detected andquantitated in vertebrate samples, preferably mammalian samples, morepreferably human samples, comprising neoplastic cells. Such samples canbe tissue specimens, tissue extracts, body fluids, cells, cell lysatesand cell extracts, among other samples. Preferred tissue specimens toassay by immunohistochemical staining, for example, include cell smears,histological sections from biopsied tissues or organs, and imprintpreparations among other tissue samples. An exemplaryimmunohistochemical staining protocol is described below in theMaterials and Methods section of Example 1. Such tissue specimens can bevariously maintained; for example, they can be fresh, frozen, orformalin-, alcohol- or acetone- or otherwise fixed and/orparaffin-embedded and deparaffinized. Biopsied tissue samples can be,for example, those samples removed by aspiration, bite, brush, cone,chorionic villus, endoscopic, excisional, incisional, needle, fineneedle, percutaneous punch, and surface biopsies, among other biopsytechniques. Preferred tissue samples are formalin-fixed,paraffin-embedded tissue samples.

Assays

Many formats can be adapted for use with the methods of the presentinvention. The detection and quantitation of CA IX protein or CA IXpolypeptide can be performed, for example, by Western blots,enzyme-linked immunosorbent assays, radioimmunoassays, competitionimmunoassays, dual antibody sandwich assays, immunohistochemicalstaining assays, agglutination assays, fluorescent immunoassays,immunoelectron and scanning microscopy using immunogold, among otherassays commonly known in the art. The quantitation of MN/CA9 geneexpression products in such assays can be adapted by conventionalmethods known in the art; for example, if the detection method is byimmunohistochemical staining, the quantitation of CA IX protein or CA IXpolypeptide can be performed by determining the percentage ofimmunoreactive cells and/or the intensity or extent of immunostaining ofimmunoreactive cells, and can additionally comprise addition ormultiplication of these values, or other mathematical calculations usingthese values.

It is also apparent to one skilled in the art of immunoassays thatantibodies to MN/CA IX proteins or polypeptides can be used to detectand quantitate MN/CA IX antigen in body tissues and/or cells ofpatients. In one embodiment, an immunometric assay may be used in whicha labelled antibody made to a MN/CA IX protein or polypeptide comprisingthe extracellular domain is used. In such an assay, the amount oflabelled antibody which complexes with the antigen-bound antibody isdirectly proportional to the amount of CA IX antigen in the sample.

The monoclonal antibodies useful according to this invention to identifyMN/CA IX proteins/polypeptides can be labeled in any conventionalmanner, for example, with enzymes such as horseradish peroxidase (HRP),fluorescent compounds, or with radioactive isotopes such as, ¹²⁵I, amongother labels. A preferred label, according to this invention is ¹²⁵I,and a preferred method of labeling the antibodies is by usingchloramine-T [47]. Also preferred is the method of labeling theantibodies using peroxidase. Many other means of visualizing the MN/CA9gene expression products known to those of skill in the art can also beused.

Exemplary Immunohistochemical Assays

An exemplary immunohistochemical assay described in Example 1 below usesantibody staining to investigate the distribution and expression patternof MN/CA IX. Paraffin-embedded tissue sections from nephrectomy patientsare deparaffinized and rehydrated, then stained with the MN/CAIX-specific monoclonal antibody M75. The sections are then reacted witha biotinylated antibody recognizing mouse IgG, and subsequently withstreptavidin-peroxidase conjugate. The immunoperoxidase complexes arethen visualized with a chromogen, such as diaminobenzidinetetrahydrochloride (DAB) or 3-amino-9-ethyl carbazole (AEC).

Nucleic Acid-Based Assays

In certain embodiments of the invention, mRNA or cDNA that encodes a CAIX protein or a CA IX polypeptide is detected and if present, thepercentage of cells in a sample that are expressing CA9 mRNA or cDNA isdetermined, and thereby correlated with a prognosis for a patient. Anexemplary nucleic acid-based method is Northern blotting, where thenucleic acid sequence used as a probe for detecting MN/CA9-specific mRNAexpression is complementary to all or part of the MN/CA9 cDNA sequence.A preparation of RNA is run on a denaturing agarose gel, and transferredto a suitable support, such as activated cellulose, nitrocellulose orglass or nylon membranes. The nucleic acids used to detect the MN/CA9mRNA or cDNA may be radiolabelled and analyzed by autoradiography.Non-radioactive labels, for example, such as fluorophores or reportergroups such as digoxigenin may also be used to detect the MN/CA9 mRNA orcDNA.

An alternate preferred method for measuring CA IX-specific mRNAexpression is the detection of CA9 mRNA expression via hybridization ofa nucleic acid probe derived from MN/CA9 cDNA sequence to RT-PCRproducts generated from RNA isolated from a biological sample. ExemplaryPCR primers designed to amplify a 240 bp cDNA fragment of the CA9 geneare sense 5′-AGGAGGATCTGCC CAGTGA-3′ [SEQ ID NO: 10]; antisense5′-GCCAATGACTCTGGTCATC-3′) [SEQ ID NO: 11.] Murakami et al. and Uemuraet al. have reported that CA IX detection by RT-PCR in patient samplescorrelate well with immunohistochemistry [10, 11].

MN Gene and Protein

The terms “MN/CA IX” and “MN/CA9” are herein considered to be synonymsfor MN. Also, the G250 antigen is considered to refer to MNprotein/polypeptide [23].

Zavada et al., WO 93/18152 and/or WO 95/34650 disclose the MN cDNAsequence [SEQ ID NO: 1], the MN amino acid sequence [SEQ ID NO: 2], andthe MN genomic sequence [SEQ ID NO: 3]. The MN gene is organized into 11exons and 10 introns.

The ORF of the MN cDNA [SEQ ID NO: 1] has the coding capacity for a 459amino acid protein with a calculated molecular weight of 49.7 kd. Theoverall amino acid composition of the MN/CA IX protein is rather acidic,and predicted to have a pI of 4.3. Analysis of native MN/CA IX proteinfrom CGL3 cells by two-dimensional electrophoresis followed byimmunoblotting has shown that in agreement with computer prediction, theMN/CA IX is an acidic protein existing in several isoelectric forms withpIs ranging from 4.7 to 6.3. [CGL3 cells are hybrid HeLa fibroblastcells that are tumorigenic, derived from HeLa D98/AH.2 (also known asHeLa S), a mutant HeLa clone that is hypoxanthine guanine phosphoribosyltransferase-deficient (HGPRT⁻) reported in Stanbridge et al., Science.215: 252-259 (15 Jan. 1982).]

The first thirty seven amino acids of the MN protein is the putative MNsignal peptide [SEQ ID NO: 6]. The MN protein has an extracellulardomain [amino acids (aa) 38414; SEQ ID NO: 7], a transmembrane domain[aa 415-434; SEQ ID NO: 8] and an intracellular domain [aa 435-459; SEQID NO: 9]. The extracellular domain contains the proteoglycan-likedomain [aa 53-111: SEQ ID NO: 4] and the carbonic anhydrase (CA) domain[aa 135-391; SEQ ID NO: 5].

The CA domain is essential for induction of anchorage independence,whereas the TM anchor and IC tail are dispensable for that biologicaleffect. The MN protein is also capable of causing plasma membraneruffling in the transfected cells and appears to participate in theirattachment to the solid support. The data evince the involvement of MNin the regulation of cell proliferation, adhesion and intercellularcommunication.

MN Proteins and Polypeptides

The phrase “MN proteins and/or polypeptides” (MN proteins/polypeptides)is herein defined to mean proteins and/or polypeptides encoded by an MNgene or fragments thereof. An exemplary and preferred MN proteinaccording to this invention has the deduced amino acid sequencerepresented by SEQ ID NO: 2. Preferred MN proteins/polypeptides arethose proteins and/or polypeptides that have substantial homology withthe MN protein [SEQ ID NO: 2]. For example, such substantiallyhomologous MN proteins/polypeptides are those that are reactive withMN-specific antibodies, preferably the Mab M75 or its equivalent. TheVU-M75 hybridoma that secretes the M75 Mab was deposited at the ATCCunder HB 11128 on Sep. 17, 1992.

A “polypeptide” or “peptide” is a chain of amino acids covalently boundby peptide linkages and is herein considered to be composed of 50 orless amino acids. A “protein” is herein defined to be a polypeptidecomposed of more than 50 amino acids. The term polypeptide encompassesthe terms peptide and oligopeptide.

As used herein, “low CA IX” or “low MN/CA IX” refers to a determinationthat 50% or less of cells in a neoplastic sample taken from an RCCpatient express the MN/CA9 gene. Correspondingly, “high CA IX” or “highMN/CA IX” refers to a determination that more than 50% of cells in aneoplastic sample taken from an RCC patient express the MN/CA9 gene.

It can be appreciated that a protein or polypeptide produced by aneoplastic cell in vivo could be altered in sequence from that producedby a tumor cell in cell culture or by a transformed cell. Thus, MNproteins and/or polypeptides which have varying amino acid sequencesincluding without limitation, amino acid substitutions, extensions,deletions, truncations, interpolations and combinations thereof, fallwithin the contemplated scope of this invention, provided the protein orpolypeptide containing them is immunogenic, and antibodies elicited bysuch a polypeptide or protein cross-react with naturally occurring MNproteins and polypeptides to a sufficient extent to provide protectiveimmunity and/or anti-tumorigenic activity when administered as avaccine. It can also be appreciated that a protein extant within bodyfluids is subject to degradative processes, such as, proteolyticprocesses; thus, MN proteins that are significantly truncated and MNpolypeptides may be found in body fluids, such as, sera. The phrase “MNantigen” is used herein to encompass MN proteins and/or polypeptides.

It will further be appreciated that the amino acid sequence of MNproteins and polypeptides can be modified by genetic techniques. One ormore amino acids can be deleted or substituted. Such amino acid changesmay not cause any measurable change in the biological activity of theprotein or polypeptide and result in proteins or polypeptides which arewithin the scope of this invention, as well as, MN muteins.

Nucleic Acid Probes

Nucleic acid probes of this invention are those comprising sequencesthat are complementary or substantially complementary to the MN cDNAsequence [SEQ ID NO: 1] or to other MN gene sequences, such as, thecomplete genomic sequence [SEQ ID NO: 3]. The phrase “substantiallycomplementary” is defined herein to have the meaning as it is wellunderstood in the art and, thus, used in the context of standardhybridization conditions. The stringency of hybridization conditions canbe adjusted to control the precision of complementarity. Two nucleicacids are, for example, substantially complementary to each other, ifthey hybridize to each other under stringent hybridization conditions.

Stringent Hybridization Conditions

Stringent hybridization conditions are considered herein to conform tostandard hybridization conditions understood in the art to be stringent.Only very closely related nt sequences having a homology of at least80-90% would hybridize to each other under stringent hybridizationconditions.

For example, it is generally understood that stringent conditionsencompass relatively low salt and/or high temperature conditions, suchas provided by 0.02 M to 0.15 M NaCl at temperatures of 50° C. to 70° C.such as, 0.15 M to 0.9 M salt at temperatures ranging from 20° C. to 55°C. Less stringent conditions can be made more stringent by addingincreasing amounts of formamide, which serves to destabilize hybridduplexes as does increased temperature, such as provided by 0.15 M to0.9 M NaCl in the presence of 50% formamide at 42° C. with a final washof 0.1% SSPE and 0.1% SDS at 65° C.

Exemplary stringent hybridization conditions are described in Sambrooket al., Molecular Cloning: A Laboratory Manual, pages 1.91 and 9.47-9.51(Second Edition, Cold Spring Harbor Laboratory Press; Cold SpringHarbor, N.Y.; 1989); Maniatis et al., Molecular Cloning: A LaboratoryManual, pages 387-389 (Cold Spring Harbor Laboratory; Cold SpringHarbor, N.Y.; 1982); Tsuchiya et al., Oral Surgery. Oral Medicine, OralPathology. 71(6): 721-725 (June 1991); and in U.S. Pat. No. 5,989,838,U.S. Pat. No. 5,972,353, U.S. Pat. No. 5,981,711, and U.S. Pat. No.6,051,226.

Antibodies

The term “antibodies” is defined herein to include not only wholeantibodies but also biologically active fragments of antibodies,preferably fragments containing the antigen binding regions. Furtherincluded in the definition of antibodies are bispecific antibodies thatare specific for MN protein and to another tissue-specific antigen.

Antibodies useful according to the methods of the invention may beprepared by conventional methodology and/or by genetic engineering.Antibody fragments may be genetically engineered, preferably from thevariable regions of the light and/or heavy chains (V_(H) and V_(L)),including the hypervariable regions, and still more preferably from boththe V_(H) and V_(L) regions. For example, the term “antibodies” as usedherein includes polyclonal and monoclonal antibodies and biologicallyactive fragments thereof including among other possibilities “univalent”antibodies [45]; Fab proteins including Fab′ and F(ab)₂ fragmentswhether covalently or non-covalently aggregated; light or heavy chainsalone, preferably variable heavy and light chain regions (V_(H) andV_(L) regions), and more preferably including the hypervariable regions[otherwise known as the complementarity determining regions (CDRs) ofthe V_(H) and V_(L) regions]; Fc proteins; “hybrid” antibodies capableof binding more than one antigen; constant-variable region chimeras;“composite” immunoglobulins with heavy and light chains of differentorigins; bispecific antibodies, preferably bispecific MAbs; “altered”antibodies with improved specificity and other characteristics asprepared by standard recombinant techniques and alsooligonucleotide-directed mutagenesis techniques [46].

The antibodies useful according to this invention to identify CA IXproteins/polypeptides can be labeled in any conventional manner, forexample, with enzymes such as horseradish peroxidase (HRP), fluorescentcompounds, or with radioactive isotopes such as, ¹²⁵I, among otherlabels. A preferred label, according to this invention is ¹²⁵I, and apreferred method of labeling the antibodies is by using chloramine-T[47].

Representative monoclonal antibodies useful according to this inventioninclude Mabs M75, MN9, MN12 and MN7 described in earlier Zavada et al.patents and patent applications. [U.S. Pat. No. 6,297,041; U.S. Pat. No.6,204,370; U.S. Pat. No. 6,093,548; U.S. Pat. No. 6,051,226; U.S. Pat.No. 6,004,535; U.S. Pat. No. 5,989,838; U.S. Pat. No. 5,981,711; U.S.Pat. No. 5,972,353; U.S. Pat. No. 5,955,075; U.S. Pat. No. 5,387,676; USApplication Nos: 20030049828 and 20020137910; and InternationalPublication No. WO 03/100029]. Monoclonal antibodies useful according tothis invention serve to identify MN proteins/polypeptides in variouslaboratory prognostic tests, for example, in clinical samples. Forexample, monoclonal antibody M75 (Mab M75) is produced by mouselymphocytic hybridoma VU-M75, which was deposited under ATCC designationHB 11128 on Sep. 17, 1992 at the American Tissue Type Culture Collection[ATCC]. The production of hybridoma VU-M75 is described in Zavada etal., International Publication No. WO 93/18152. Mab M75 recognizes boththe nonglycosylated GST-MN fusion protein and native CA IX protein asexpressed in CGL3 cells equally well. The M75 Mab recognizes both nativeand denatured forms of the CA IX protein [14].

General texts describing additional molecular biological techniquesuseful herein, including the preparation of antibodies include Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymoloqy, Vol. 152, Academic Press, Inc., Sambrook et al., MolecularCloning: A Laboratory Manual, (Second Edition, Cold Spring HarborLaboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3; CurrentProtocols in Molecular Biology, F. M. Ausabel et al. [Eds.], CurrentProtocols, a joint venture between Green Publishing Associates, Inc. andJohn Wiley & Sons, Inc. (supplemented through 2000), Harlow et al.,Monoclonal Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988), Paul [Ed.]; Fundamental Immunology, LippincottWilliams & Wilkins (1998), and Harlow et al., Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press (1998).

MN/CA IX and Prognosis: Use in RCC Therapy Selection

As low levels of CA IX staining correlated with worse outcome (Example1), detection and quantitation of CA IX can be used to identifyhigh-risk patients in need of aggressive therapies, including adjuvantimmunotherapy and CA9-targeted therapies. Careful patient selection andstratification to various adjuvant immunotherapies may delineate thosepatients most likely to respond to treatment. Responses to systemiccytokine therapy, for example with IFN or IL-2, among other cytokines,in metastatic RCC have been promising. Preliminary data from Bui et al.[15] suggest a relationship between CA IX and immunotherapy response.Similarly, therapies based on monoclonal antibodies to CA IX orimmunotherapy with CA IX-based RCC vaccine [15, 19-21], or CA9-directedgene therapies can also be considered according to CA IX detection andquantitation.

In addition, the prognostic methods of the invention can use CA IXdetection and quantitation in combination with conventional markers,such as tumor grade and/or tumor stage. The immunohistochemical analysisdisclosed herein revealed that lowest survival rates are seen when thereis low CA IX in high grade tumors, and low CA IX in high stage tumors.Such patients would be candidates for more aggressive therapy regimens.

Therapeutic Use of MN-Specific Antibodies: The MN-specific antibodies,monoclonal and/or polyclonal, preferably monoclonal, may be usedtherapeutically in the treatment of CA9-expressing renal cell carcinoma,either alone or in combination with chemotherapeutic drugs or toxicagents, such as ricin A. Further preferred for therapeutic use would bebiologically active antibody fragments. Also preferred MN-specificantibodies for such therapeutic uses would be MN-specific humanizedmonoclonal antibodies, fully human monoclonal antibodies, and/orbispecific antibodies.

MN-specific antibodies can be administered in a therapeuticallyeffective amount, preferably dispersed in a physiologically acceptable,nontoxic liquid vehicle, to patients afflicted with renal cell carcinomaexpressing MN/CA IX. The MN-specific antibody can be given alone or as acarrier of an anti-tumor drug. Among the various antiproliferative,antineoplastic or cytotoxic agents that may be linked to the MN-specificantibodies are antimetabolites, such as the antifolate, methotrexate, orthe purine or pyrimidine analogs mercaptopurine and fluorouracil. Othersinclude antibiotics, lectins such as ricin and abrin, toxins such as thesubunit of diphtheria toxin, radionuclides such as ²¹¹Astatine and¹³¹Iodine, radiosensitizers such as misanidazole or neutron sensitizerssuch as boron containing organics. Such agents may be attached to theantibody by conventional techniques such as glutaraldehydecross-linking.

MN-specific antibodies can be used to target cytotoxic cells (e.g. humanT cells, monocytes or NK cells). Cytotoxic cells can be attached toMN-expressing tumor cells through Fc receptors on the cytotoxic cells,which bind the Fc portion of a MN-specific antibody, or via a bridgingantibody of dual specificity, that is, a bispecific antibody specificfor MN protein and for the cytotoxic cell.

The cytotoxic cell can be targeted by allowing the bispecific antibodyto bind the cell. After targeting, the cells can be administered to thepatient. Therapy with targeted cells can be used as an adjunct tosurgical therapy, radiation therapy, or chemotherapy.

Anti-Idiotype MN-Specific Antibodies as Tumor Vaccines, andAnti-Anti-Idiotype Antibody Sera as Immunotherapeutic: MN-specificanti-idiotype antibodies have therapeutic utility as a vaccine forneoplastic disease associated with abnormal MN expression. MN-specificanti-anti-idiotype sera also have therapeutic anti-tumor efficacy. Thosetherapeutic utilities are demonstrated by research done with theMN-specific G250 MAb, and anti-idiotype antibodies thereto (Ab2), andfurther anti-anti-idiotype sera (Ab3) as demonstrated by the studiesdescribed below.

Uemura et al., Biotherapy (Japan) 10(3): 241-244 (1996) (Englishsummary) define an anti-idiotype antibody (Ab2) as “an antibody directedagainst an antigenic determinant located within a variable region of theimmunoglobulin molecule. Ab2 mimicking the normal antigen (so-calledinternal image Ab2) may be used as a surrogate antigen for vaccinationto trigger the host's immune system specifically against the nominalantigen.”

Uemura et al., id., having previously isolated six internal image murineAb2s directed against the G250 MAb—NUH31, 51, 71, 82 (IgG1) and NUH44(IgG2a), explores the application of monoclonal Ab2 as tumor vaccines.Uemura et al. investigated in view of “previous results that RCCtumor-associated-antigen-related idiotype vaccination inducedantigen-specific humoral as well as cellular responses, the antitumorefficacy of anti-anti-idiotype antibody (Ab3) sera obtained from miceimmunized with different internal image Ab2 that . . . mimic theRCC-associated antigen . . . G250 [MN] . . . Nu/nu BALB/c mice carryingsmall established NU12 human RCC xenografts (G250+, 20 mm³) . . .receiving an s.c. injection of 2×10⁵ SK-RC-52 (G250+) RCC cells weretreated by i.p. injection of 0.2 ml Ab3 sera. This treatment resulted incomplete tumor rejection and significant tumor growth inhibition ascompared to control groups (p<0.01).” Uemura et al. concluded that“immunization with Ab2s elicits powerful anti-tumor effects inimmunocompetent animals.”

Uemura et al., J. Urol. 159(5)(Suppl.): Abstract 724 (May 1998),describe MN as an immunotherapeutic target for renal cell carcinoma(RCC). The therapeutic potential of the MN-specific MAb G250 wasevaluated in combination with IFN/IL-2/MCSF (interferon, interleukin-2,macrophage colony stimulating factor) and Ab2 (NUH82)-induced mouseserum (Ab3-82). Ab2s are monoclonal anti-idiotype antibodies raisedagainst MAbG250 which have been shown to be useful as tumor vaccines forRCC.

Uemura et al., id. reported that mice with NUR-2 RCC xenografts weretreated by peri-tumor injection of MAbG250 and/or cytokines or 0.2 ml ofAb3 sera with/without MCSF. The tumor volume in MAbG250 treated animalswas significantly lower than in the controls. IFN or IL-2 treatments wassimilarly effective, but MCSF resulted in no significant tumorinhibition. The IFN/IL-2/MAbG250 therapy increased significantly theanti-tumor effects as compared to MAbG250 or cytokine monotherapy.Further, Ab3-based (Ab2-induced) immunotherapy resulted in tremendoustumor monotherapy growth inhibition as compared to MAbG250 or the othercytokine combination therapies.

Gene Therapy: Further, a CA IX-specific molecule, such as an anti-CA IXmonoclonal antibody, could be coupled to a vector for targeted deliveryto CA IX-specific expressing cells for gene therapy (for example, withthe wild-type von Hippel-Lindau gene), or for effecting the expressionof cytotoxic proteins.

Such methods can be of particular diagnostic and prognostic importancebecause MN/CA9 is a hypoxia-regulated gene. Hypoxia combined with CA IXoverepression indicates that the mammal from whom the sample was takenis considered to have a poorer prognosis, and decisions on treatment forsaid mammal are made in view of the presence of said hypoxic conditions.MN/CA IX as a hypoxia marker is useful in general in making therapeuticdecisions. For example, a cancer patient whose tumor is known to expressMN/CA IX at an abnormally high level would not be a candidate forcertain kinds of chemotherapy and radiotherapy, but would be a candidatefor hypoxia-selective chemotherapy.

Brown [48] points out at page 157 that “solid tumours are considerablyless well oxygenated than normal tissues. This leads to resistance toradiotherapy and anticancer chemotherapy, as well as predisposing toincreased tumour metastases.” Brown explains how tumor hypoxia can beexploited in cancer treatment. One strategy to exploit tumor hypoxia forcancer treatment proposed by Brown [48] is to use drugs that are toxiconly under hypoxic conditions. Exemplary and preferred drugs that couldbe used under that strategy include tirapazamine and AQ4N, a di-N-oxideanalogue of mitozantrome.

A second mode of exploiting hypoxia proposed by Brown [48] is by genetherapy strategies developed to take advantage of the selectiveinduction of HIF-1. Preferably, a gene therapy vector referred to abovecomprises a MN/CA IX promoter or MN/CA9 promoter fragment comprising theMN/CA IX hypoxia response element (HRE) or a HRE of another gene, andmore preferably wherein the CA IX promoter or CA IX promotor fragmentcomprises more than one HRE, wherein said HRE or HREs is or are eitherof MN/CA9, and/or of other genes and/or of genetically engineered HREconsensus sequences in a preferred context.

Brown notes that a tumor-specific delivery system can be developedwherein a promoter that is highly responsive to HIF-1 would drive theexpression of a conditionally lethal gene under hypoxic but not normoxicconditions. The MN/CA9 promoter is just such a promoter highlyresponsive to hypoxia, as well as MN/CA9 promoter fragments comprisingone or more HREs. “Expression of an enzyme not normally found in thehuman body could, under the control of a hypoxia-responsive promoter[the MN/CA9 promoter], convert a nontoxic pro-drug into a toxic drug inthe tumour.” [Brown [48], page 160.] Exemplary is the use of thebacterial cytosine deaminase, which converts the nontoxic5-fluorocytosine to the anticancer drug 5-fluorouracil (5FU) cited byBrown to Trinh et al. [49].

Ratcliffe et al., U.S. Pat. Nos. 5,942,434 and 6,265,390 explain howanti-cancer drugs become activated under hypoxia, but that the use of adrug activation system, wherein the enzyme that activates the drug issignificantly increased under hypoxia, results in much enhancedtherapeutic effect.

Computerized MN/CA IX Data Analysis: The correlation of MN/CA9expression (for example, level and/or extent) and RCC prognosis can beanalyzed by any number of methods known to one of skill in the art, forexample, by computer program. Such a computer program could comprisealgorithms for correlating CA9 expression data derived from a renal cellcarcinoma patient with a probable prognosis.

The following examples are for purposes of illustration only and are notmeant to limit the invention in any way.

Example 1

The following experiments were designed to investigate whether MN/CA IXcould be used as a prognostic marker, as well as a diagnostic marker inRCC, particularly CCC. Nephrectomy specimens from 92 patients were usedin this study. Eighty (80) of these were renal cell carcinomas, 10adenomas and 2 oncocytomas. Of the renal cell carcinomas, 67 were clearcell carcinomas (CCCs). Immunohistochemical analysis using the MN/CAIX-specific monoclonal antibody (M75) was performed on paraffin embeddedspecimens. MN/CA IX staining was correlated with tumor stage, grade,lymph node involvement, distant metastasis and cumulative survival time.

MN/CA IX was present in 91.2% of the clear cell carcinomas. Low stainingwas a poor prognostic factor, and conversely high staining a goodprognostic factor. MN expression was found to be the best prognosticfactor when compared with T stage and grade. Even in low-grade and stagetumors, the presence of low MN expression correlated with loweredsurvival times.

The conclusion from the study disclosed herein is that MN is asignificant molecular marker in RCCs, particularly CCCs. Decreased MNexpression is independently associated with poor survival. MN can beused to predict clinical outcome and identify high-risk patients in needof adjuvant immunotherapy and MN targeted therapies.

Material and Methods

Samples of nephrectomy specimens from 80 cases of RCC, ten renaladenomas and two renal oncocytomas, obtained from the archives of thePathology Department of çukurova University, were evaluated. Allpatients had been operated upon in the Urology Department of the sameuniversity hospital between 1996 and 2003. Histologic slides of eachcase were reviewed for diagnostic reassessment. Grading was performedaccording to Fuhrman nuclear grading system [13].

5-μm thick sections of formalin-fixed, paraffin-embedded tissue sampleswere deparaffinized and rehydrated through a series of graded alcohols.Antigen retrieval was carried out in citrate buffer (pH 6) for 10minutes in a microwave oven. For 20-25 minutes, they were rested to cooldown to room temperature. After washing slides in PBS for 5 minutes,incubation was done with blocking serum for 10 minutes. Then, a mousemonoclonal antihuman antibody (M75) raised to external domain of MN/CAIX was applied at a concentration of 1/300 for 60 minutes at roomtemperature [14]. After washing the slides for 5 minutes in PBS, theywere incubated at 25° C. in ready-to-use biotinylated UniversalSecondary Antibody for 20 minutes (NCL-RTU-D, Novacastra, UK).Afterwards, the slides were washed in PBS for 5 minutes and thenincubated at 25° C. in RTU streptavidin/peroxidase complex reagent for10 minutes. After incubation, slides were washed twice with PBS for 5minutes once again. The immunperoxidase was visualized with AEC(3-amino-9-ethylcarbazole; DAKO, USA). The sections were counterstainedwith Mayer's hematoxylin and then coverslipped. An isotypic antibody wasused in all staining procedures as a negative control.

The immunohistochemical results were scored semiquantitatively, basedupon the percentage of positive cells seen in a total field of asingle-section. MN/CA IX antigen is a membrane-associated protein.Therefore, when a cell exhibited sharp and clear membrane staining, itwas interpreted as MN/CA IX immunoreactive. Only membrane stainingpattern was evaluated using a 0 to 2+ scale (0, completely negative, 1+focal when ≦50% of the cells stained, 2+ diffuse when >50% of the cellsstained as described by Liao et al. [12]). The pattern of staining wasconsidered as of low and high expression when the intensity is 0 to 1+and 2+, respectively. All specimens were examined by two blindedpathologists. Interobserver reliability between the two was 88%.

SPSS (“Statistical Package for the Social Sciences,” originally) forWindows version 10.0 was used for statistical analyses. Differences inMN/CA IX staining levels between independent groups were evaluated byChi square or Fisher's exact test. The Kaplan-Meier method was used toestimate cumulative survival and log-rank test was applied to comparestratified survival functions. The Cox proportional hazards model wasused to test the statistical independence and significance of MN/CA IX(age, grade, T stage and MN/CA IX were used in the model as dependentvariables). Data were expressed as mean±SD (standard deviation), n(number of cases) and percent (%). A p value less than 0.05 wasconsidered significant.

Results

Of the total 92 specimens, 80 were RCC and of the RCCs 67 clear cellsubtype (CCC), 10 papillary and 3 chromophobe. The remaining 12consisted of 10 adenomas and 2 oncocytomas.

Of the 80 RCCs, 10, 6 of which were CCCs, did not show any staining atall. Therefore MN expression in RCC tumor specimens was 87.5%. Of thosewhich showed staining, 31 stained less than 50% and of those 25 wereCCCs. 39 RCCs, 36 CCC and 3 papillary showed more than 50% staining.[Exemplary immunohistochemical staining showed a high pattern of MN/CAIX staining in papillary carcinoma (FIG. 1), and intensive membraneousstaining in clear cell carcinoma (FIG. 2).] None of the 10 adenomasshowed staining. Of the 2 oncocytomas, 1 did not stain, the other showedless than 50% staining (Table 1).

TABLE 1 Expression of the MN/CA IX in RCC and benign renal lesionsPattern of Staining n None <50% >50% Renal Cell Carcinoma Clear CellCarcinoma 67 6 25  36 Papillary Cell Carcinoma 10 2 5  3 ChromophobeCell 3 2 1 — Adenoma 10 10 — — Oncocytoma 2 1 1 —

Overall, 67 RCC patients with clear cell type were included in theanalysis. The patients' age at the time of surgery ranged from 18 to 81years (mean of 54.8±11.6 years). The male/female ratio was 1.9:1 with 44male patients (65.7%) and 23 female patients (34.3%). Radicalnephrectomy was performed on 62 patients (92.5%) and partial nephrectomyon 5 (7.5%). As for pathologic stage, 22 cases were stage I, 31 stageII, 8 stage III and 6 were stage IV. As for pathological grade, therewere 24 grade 1 (35.8%), 20 grade 2 (29.9%), 16 grade 3 (23.9%) and 7grade 4 cases (10.4%). Table 2 lists patient and tumor characteristics.

TABLE 2 Characteristics of RCC patients Patient Data PatientCharacteristics n = 67 (%) Sex Male 44 (65.7) Female 23 (34.3) SideRight 31 (46.3) Left 36 (53.7) Age (years) Mean ± SD 54.8 ± 11.6 Median54.0 Min-Max 18-81 Operation Partial nephrectomy 5 (7.5) Radicalnephrectomy 62 (92.5) T Stage 1 22 (32.8) 2 31 (46.2) 3 8 (11.9) 4 6(8.9) Grade 1 24 (35.8) 2 20 (29.9) 3 16 (23.9) 4 7 (10.4) MetastasisYes 4 (6.0%) No 63 (94.0%)

The relation of staining pattern in CCC with pathologic T and N stage, Mstage and grade of the tumor were evaluated, and the results are givenin Table 3. Low stage T tumors showed high CA IX staining in 66% of thecases, whereas high stage T tumors showed mostly low CA IX staining(92.9%). When there was no lymph node involvement, there was a highpattern of staining in 57.1% of the cases; whereas with lymph nodeinvolvement, a low pattern of CA IX staining was seen in all (100%)cases. Of the total, 63 patients had no distant metastasis; 36 (57.1%)of them showed a high pattern of CA IX staining. All of the 4 (100%)patients with distant metastasis had a low pattern of CA IX staining.Low grade (1 or 2) tumors had mostly high CA IX staining (71.1%);whereas high grade (3 or 4) tumors mostly had low CA IX staining (81.8%)(Table 3).

TABLE 3 Relation of staining pattern with pathologic stage T and N,metastasis and grade Pattern of staining low (n = 31) high (n = 36) n(%) n (%) p value T-Stage Low (1 or 2) 18 (34.0) 35 (66)   0.000 High (3or 4) 13 (92.9) 1 (7.1) Nodes No 27 (42.9) 36 (57.1) 0.04 Yes  4 (100.0)— Metastasis No 27 (42.9) 36 (57.1) 0.04 Yes  4 (100.0) — Grade Low (1or 2) 13 (28.9) 32 (71.1) 0.000 High (3 or 4) 18 (81.8)  4 (18.2)

Of the CCC patients, 6 were lost to follow-up. Mean and mediancumulative survival of the 61 remaining patients according to T stage,grade and CA IX expression is given in detail in Table 4 and FIGS. 3 a,b, c, 4 and 5. In the group with high CA IX expression, mean cumulativesurvival was 64.1 months. When grade was considered, in the low gradegroup mean cumulative survival was 53.5 months; however, when there waslow CA IX expression in the low-grade tumors, survival dropped to 28.7months. Similarly, the cumulative survival rate of 49.4 months in thelow T stage tumors dropped to 28.7 months when such low grade tumorsshowed a low level of CA IX staining. Cumulative survival correlatedwith the level of CA IX staining (p=0.000) with grade (p=0.006) and Tstage (p=0.02) of the tumor.

TABLE 4 Mean and median cumulative survival (months) for patients (n =61) according to T Stage, Grade and MN/CA IX expression Mean (median)dead/total p value Grade Low (1 or 2) 53.5 (61) 20/39 0.006 High (3 or4) 22.6 (16) 17/22 T stage Low (1 or 2) 49.4 (39) 26/47 0.02 High (3 or4) 19.3 (12) 11/14 CA IX Low (<%50) 23.4 (12) 24/29 0.000 High (>%50)64.1 (64) 13/32 Grade (Gr) and CA IX Low Gr-Low CA IX 28.7 (11)  8/110.0003 Low Gr-High CA IX 61.7 (67) 12/28 High Gr-Low CA IX 15.5 (14)16/18 High Gr-High CA IX 61.0 (40) 1/4 T stage (Stg) and CA IX Low TStg-Low CA IX 19.7 (11) 14/16 0.0001 Low T Stg-High CA IX 64.3 (64)12/31 High T Stg-Low CA IX 20.2 (16) 10/13 High T Stg-High CA IX   8 (—)1/1

Conversely, the high tumor grade patients had a survival rate of 22.6months, but when CA IX staining was high in the high grade tumors,survival jumped up to 61 months. A similar conclusion could not bearrived at in the high stage-high CA IX expression group because therewas only one patient, and he survived for 8 months.

The Cox proportional hazards model results are shown in Table 5. Theresults show that CA IX is an independent and significant factor relatedwith survival rate in RCC patients.

TABLE 5 Results of Cox regression model Variables in 95% CI for ORequation OR (Lower-Upper) P value Age 0.9 (0.9-1.0) 0.270 Grade 1.5(0.7-3.3) 0.304 T Stage 1.3 (0.6-3.1) 0.529 CA IX 3.9 (1.7-9.0) 0.001

Discussion

In this study of 80 RCCs, CA IX staining was 87.5%, which is consistentwith previous reports [7]. In the CCC group, the staining rate was 91%,which suggests CA IX to be a strong biomarker especially for the clearcell subtype of RCC. Murakami et.al. [10] found CA IX expression in CCCto be 92%; that rate was reported by Bui et al. [15] to be 94%.

As for its prognostic value, a low level of CA IX staining correlatedwith worse outcome. High T stage tumors had low CA IX expression(92.9%). Even though only four of the cases had lymph node involvement,all of those cases had a low level of CA IX staining. As for distantmetastasis, all had low CA IX expression again, whereas only 42.9% ofcases with no metastasis stained in the same pattern. Again with hightumor grade, there was a high rate of low CA IX expression (81.8%). Whenthe cumulative survival rate was correlated with the tumor grade, stageand CA IX expression, CA IX expression was at least as good a factoreffecting survival rate as T-stage and tumor grade. Since the number ofpatients was insufficient, survival due to N and M stages could not beestimated.

The overall survival rate in the subject study group is lower than thatreported in the literature [1]. That again may be due to the limitednumber as well as possibly the socioeconomic status of the patients.

In all of the low CA IX, high stage and high tumor grade groups,survival rates were similar (19.3, 22.6, 23.6 months, respectively).However, lowest survival rates were seen when there is low CA IX in highgrade (15.5 months) and low CA IX in high stage (20.3 months) tumors. Itis therefore crucial to predict those subsets of patients in order toestablish more aggressive therapy regimens.

RCC has been shown to respond to biological immunotherapy. Responses tosystemic cytokine therapy in metastatic RCC are promising, although theoverall results are still inadequate. Careful patient selection andstratification to various adjuvant immunotherapies may delineate thosepatients most likely to respond to treatment.

In conclusion, MN/CA IX is shown herein to be a significant andpromising molecular marker in the prognosis of RCCs/CCCs. DecreasedMN/CA IX expression is independently associated with poor survival.MN/CA IX can be used to predict clinical outcome and tumor behavior.MN/CA IX can identify high-risk patients who could benefit from adjuvantimmunotherapy, and MN/CA IX targeted therapies among other therapies, asexemplified by those described above.

BUDAPEST TREATY DEPOSITS

The materials listed below were deposited with the American Type CultureCollection (ATCC) now at 10810 University Blvd., Manassus, Va.20110-2209 (USA). The deposits were made under the provisions of theBudapest Treaty on the International Recognition of DepositedMicroorganisms for the Purposes of Patent Procedure and Regulationsthereunder (Budapest Treaty). Maintenance of a viable culture is assuredfor thirty years from the date of deposit. The hybridomas and plasmidswill be made available by the ATCC under the terms of the BudapestTreaty, and subject to an agreement between the Applicants and the ATCCwhich assures unrestricted availability of the deposited hybridomas andplasmids to the public upon the granting of patent from the instantapplication. Availability of the deposited strain is not to be construedas a license to practice the invention in contravention of the rightsgranted under the authority of any Government in accordance with itspatent laws.

Deposit Date ATCC # Hybridoma VU-M75 Sep. 17, 1992 HB 11128 MN 12.2.2Jun. 9, 1994 HB 11647 Plasmid A4a Jun. 6, 1995 97199 XE1 Jun. 6, 199597200 XE3 Jun. 6, 1995 97198

Similarly, the hybridoma cell line V/10-VU which produces the V/10monoclonal antibodies was deposited on Feb. 19, 2003 under the BudapestTreaty at the International Depository Authority (IDA) of the BelgianCoordinated Collections of Microorganisms (BCCM) at the Laboratoriumvoor Moleculaire Biologie-Plasmidencollectie (LMBP) at the UniverseitGent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium [BCCM/LMBP] underthe Accession No. LMBP 6009CB.

The description of the foregoing embodiments of the invention have beenpresented for purposes of illustration and description. They are notintended to be exhaustive or to limit the invention to the precise formdisclosed, and obviously many modifications and variations are possiblein light of the above teachings. The embodiments were chosen anddescribed in order to explain the principles of the invention and itspractical application to enable thereby others skilled in the art toutilize the invention in various embodiments and with variousmodifications as are suited to the particular use contemplated.

All references cited herein are hereby incorporated by reference.

1. A method which is prognostic for renal cell carcinoma afflicting avertebrate, said method comprising: (a) detecting the presence orabsence of MN/CA9 gene expression product in a sample comprisingneoplastic cells taken from said vertebrate, (b) if MN/CA9 geneexpression product is present in said sample, quantitating the leveland/or extent of said MN/CA9 gene expression product relative to thenumber of cells in said sample, and (c) determining that said vertebratehas a poorer prognosis if the level and/or extent of MN/CA9 geneexpression product of steps (a) and (b) indicates that 50% or fewer ofcells in said sample express MN/CA9 gene expression product; whereinsaid MN/CA9 gene expression product is encoded by a nucleotide sequenceselected from the group consisting of: (2) SEQ ID NO: 1's coding region;(2) nucleotide sequences that hybridize under stringent hybridizationconditions of 50% formamide at 42 degree C. to complement of SEQ ID NO:1's coding region; and (3) nucleotide sequences that differ from SEQ IDNO: 1's coding region or from the nucleotide sequences of (2) in codonsequence due to the degeneracy of the genetic code.
 2. The method ofclaim 1, wherein said renal cell carcinoma in said vertebrate isselected from the group consisting of renal clear cell carcinoma,papillary cell carcinoma and chromophobe carcinoma.
 3. The method ofclaim 1 wherein said renal cell carcinoma is renal clear cell carcinoma.4. The method of claim 1, wherein detecting step (a) comprises the useof immunohistochemical staining with MN/CA IX-specific antibody todetect the presence or absence of MN/CA IX protein in the sample, andwherein quantitating step (b) comprises determining the percentage ofMN/CA IX immunoreactive cells, wherein if 50% or fewer of cells in saidsample are immunoreactive, concluding in step (c) that said vertebratehas a poorer prognosis than if more than 50% of cells in said sample areimmunoreactive.
 5. The method of claim 1, wherein detecting step (a)comprises the use of immunohistochemical staining with MN/CA IX-specificantibody to detect the presence or absence of MN/CA IX protein in thesample, and wherein quantitating step (b) comprises determining a MN/CAIX immunoreactivity score of cells in said sample, comprisingdetermining the percentage of immunoreactive cells with cell membranestaining, wherein the percentage of immunoreactive cells is assigned animmunoreactivity score with a value of 0 if no immunoreactive cells, avalue of 1 if 50% or less immunoreactive cells, or a value of 2 if morethan 50% immunoreactive cells; and wherein if the immunoreactivity scoreof the sample is 1 or less, concluding in step (c) that said vertebratehas a poorer prognosis than if said immunoreactivity score is
 2. 6. Themethod of claim 1, wherein a poorer prognosis is measured in terms ofshortened cumulative survival, increased risk of recurrence of saidrenal cell carcinoma and/or increased risk of metastasis.
 7. The methodof claim 1, wherein said sample is a formalin-fixed, paraffin-embeddedtissue sample.
 8. The method of claim 1, wherein said MN/CA9 geneexpression product comprises an MN/CA IX protein or MN/CA IXpolypeptide.
 9. The method of claim 1, wherein said MN/CA9 geneexpression product comprises a mRNA encoding an MN/CA IX protein orMN/CA IX polypeptide, or a cDNA complementary to said mRNA.
 10. Themethod according to claim 1, wherein said detecting step (a) comprisesthe use of an assay selected from the group consisting of Western blots,enzyme-inked immunosorbent assays, radioimmunoassays, competitionimmunoassays, dual antibody sandwich assays, immunohistochemicalstaining assays, agglutination assays, and fluorescent immunoassays. 11.The method of claim 1, wherein said detecting step (a) is by in situhybridization, Northern blotting, PCR, RT-PCR, real-time PCR, or byquantitative real-time RT-PCR.
 12. The method according to claim 1,wherein said detecting step (a) comprises the use of the monoclonalantibody secreted by the hybridoma VU-M75 which has Accession No. ATCCHB
 11128. 13. The method according to claim 1, wherein said detectingstep (a) is by immunohistochemical staining, and wherein saidquantitating step (b) comprises determining the percentage of MN/CA IXimmunoreactive cells.
 14. The method according to claim 1, wherein saiddetecting step (a) is by immunohistochemical staining, and wherein saidquantitating step (b) comprises determining the percentage and/or theintensity and/or extent of immunostaining of immunoreactive cells. 15.The method of claim 1, wherein said vertebrate is a mammal.
 16. Themethod of claim 15, wherein said mammal is a human.
 17. The method ofclaim 1, wherein said prognostic method is used as an aid in theselection of treatment for said renal cell carcinoma afflicting saidvertebrate.
 18. The method of claim 17, wherein if quantitating step (b)indicates that more than 50% of cells in said sample express MN/CA9 geneexpression product, said vertebrate is identified as a candidate forMN/CA9-directed therapy.
 19. The method of claim 1, wherein even if thetumor T-stage is 3 or higher and/or the Fuhrman grade is 3 or higher insaid vertebrate, if more than 50% of cells in said sample express MN/CA9gene expression product, said vertebrate has a better prognosis than if50% or less of cells in said sample express MN/CA9 gene expressionproduct.
 20. The method of claim 1, wherein even if the tumor T-stage is2 or lower and/or the Fuhrman grade is 2 or lower in said vertebrate, if50% or less of cells in said sample express MN/CA9 gene expressionproduct, said vertebrate has a worse prognosis than if more than 50% ofcells in said sample express MN/CA9 gene expression product.
 21. Themethod of claim 17, wherein if said renal cell carcinoma has a T-stageof 3 or higher and/or a Fuhrman grade of 3 or higher, then if thequantitating step (b) indicates that 50% or less of cells in said sampleexpress MN/CA9 gene expression product, said vertebrate should betreated with more aggressive therapy regimens.